Insulin is composed of 51 amino acids, and has a molecular weight of 5808 dalton (Da). Insulin is produced in the beta cells of islets of Langerhans in the pancreas, and is stored as a hexamer (a unit of six insulin molecules) before being absorbed into the blood vessel in a biologically active monomeric form. The hexamer formation is facilitated by the coordination of zinc ions and hydrophobic interaction between three dimers. Within the hexamer, two metal ion-binding sites exist and three histidine residues derived from the three dimers are involved in each site. The binding sites exist at both ends of the hexamer or at the bottom of tunnel structure through the center from the surface according to the structural state of the insulin hexamer (T or R state).
Currently, most commercial recombinant insulin and insulin analogues exist as hexamer formulations. That is, they are formulated by including 3 mg/mL or more of insulin in a buffer solution containing the hexamer-stabilizing compounds, zinc and phenol (or cresol). Compared to the monomeric forms, the hexamer formulations provide excellent resistance to fibrillation and deamination, thereby improving the stability of insulin and extending the expiration date. Moreover, after subcutaneous injection, the hexamer formulations show a slower absorption from the injection site into the blood than the monomeric form, and thus they have the advantage of sustained duration of action. According to the previous studies, the slow absorption rate is explained by an inverse relation between the molecular size and capillary permeability at the depot. These properties of hexamers were applied to the recently developed long-acting insulin analogues to cause a delayed or sustained absorption of insulin after subcutaneous injection. A representative example is an insulin detemir prepared by the attachment of a fatty acid chain to a lysine at position 29 on the B chain of native insulin (Havelund et al., 2004). According to this study, while insulin detemir injected forms in the body a dihexamer, it forms a large molecular complex by hydrophobic interaction with albumin. Thus, subcutaneous half-life, which is a time taken by half of the drug injected subcutaneously to pass through the capillary wall, was 4 times longer than the native insulin hexamer.
However, the hexamer formulations are disadvantageous in that they cannot be applied to insulin analogues that have modification at the first amino acid phenylalanine on the B chain of native insulin, because the phenylalanine residue is involved in the structural stability of hexamer. According to the previous study using PEGylated insulin (Hinds, et al., 2000), when insulin analogues prepared by attachment of 750 Da or 2,000 Da-sized PEG to the amino terminus of the B chain of native insulin were analyzed by UV-circular dichroism and sedimentation equilibrium, most of them existed as a monomer within the concentration range of 0.1-1.0 mM. On the contrary, native insulin mostly exists as a hexamer within the corresponding concentration range. Thus, it is difficult to have the advantage of sustained absorption of insulin after subcutaneous injection using the formulated PEGylated insulin hexamer. Other examples of the insulin analogues are albumin-insulin conjugate, glycosylated insulin or the like.
Therefore, there is a urgent need to develop a formulation that induces multimer formation of insulin analogues for the improvement of their pharmacological properties such as stability and sustainability.